ATP-specificity of succinyl-CoA synthetase from Blastocystis hominis.
Acta Crystallogr D Struct Biol
; 75(Pt 7): 647-659, 2019 Jul 01.
Article
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| MEDLINE
| ID: mdl-31282474
ABSTRACT
Succinyl-CoA synthetase (SCS) catalyzes the only step of the tricarboxylic acid cycle that leads to substrate-level phosphorylation. Some forms of SCS are specific for ADP/ATP or for GDP/GTP, while others can bind all of these nucleotides, generally with different affinities. The theory of `gatekeeper' residues has been proposed to explain the nucleotide-specificity. Gatekeeper residues lie outside the binding site and create specific electrostatic interactions with incoming nucleotides to determine whether the nucleotides can enter the binding site. To test this theory, the crystal structure of the nucleotide-binding domain in complex with Mg2+-ADP was determined, as well as the structures of four proteins with single mutations, K46ßE, K114ßD, V113ßL and L227ßF, and one with two mutations, K46ßE/K114ßD. The crystal structures show that the enzyme is specific for ADP/ATP because of interactions between the nucleotide and the binding site. Nucleotide-specificity is provided by hydrogen-bonding interactions between the adenine base and Gln20ß, Gly111ß and Val113ß. The O atom of the side chain of Gln20ß interacts with N6 of ADP, while the side-chain N atom interacts with the carbonyl O atom of Gly111ß. It is the different conformations of the backbone at Gln20ß, of the side chain of Gln20ß and of the linker that make the enzyme ATP-specific. This linker connects the two subdomains of the ATP-grasp fold and interacts differently with adenine and guanine bases. The mutant proteins have similar conformations, although the L227ßF mutant shows structural changes that disrupt the binding site for the magnesium ion. Although the K46ßE/K114ßD double mutant of Blastocystis hominis SCS binds GTP better than ATP according to kinetic assays, only the complex with Mg2+-ADP was obtained.
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MEDLINE
Assunto principal:
Succinato-CoA Ligases
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Modelos Moleculares
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Trifosfato de Adenosina
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Blastocystis hominis
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article